Project 1: Vinculin stabilizes nascent adhesions and establishes lamellipodium-lamella border in migrating cells Ingo Thievessen, R. Ross The actin cytoskeleton at the leading edge of migrating cells consists of two actin networks, the lamellipodium (LP), characterized by fast polymerization-driven retrograde actin flow and the lamellum (LM) with slow myosin II (myoII) mediated actin flow. The engagement of LP actin to the ECM via nascent integrin-mediated focal adhesions (FA) establishes the flow velocity gradient between LP and LM. Nascent adhesions then elongate and mature via myoII LM actin flow. How integrins are connected to the retrograde actin flow is not known. Using primary murine embryonic fibroblasts (MEF) deficient for the vinculin gene (Vcl), we sought to test the hypothesis that vinculin mediates the coupling of actin retrograde flow to the ECM in FA. Our results suggest that vinculin stabilizes nascent FA by coupling to lamellipodial actin flow, thus establishing the flow velocity gradient between LP and LM and promoting the maturation of nascent adhesions. This implicates vinculin as an essential component in linking the dynamic actin cytoskeleton to the ECM during cell migration. This work was done in collaboration with Bob Ross, and has been presented at Cell Biology Society meeting and Gordon Conference and a manuscript describing these results was published in the JCB this past yearProject 2: Nanoscale architecture of integrin-based cell adhesions Project 2:Specific protein-interactions regulate the three-dimensional nanoscale organization of vinculin within focal adhesions L.B. Case, G. Shtengel, H.F. Hess, S. Campbell C.M. Waterman Specific protein-interactions regulate the three-dimensional nanoscale organization and conformation of vinculin within focal adhesions. L.B. Case, G. Shtengel, M. Baird, M. Davidson, S. Campbell, H.F. Hess, C.M. Waterman Previous studies have shown that FAs have a conserved stratified nanoscale structure, with an integrin signaling layer (ISL) 10-20 nm from the plasma membrane, an actin regulatory layer (ARL) 100 nm from the membrane that extends into the stress fiber, and a force transduction layer (FTL) that spans these two layers. Vinculin is an FA protein that functions in signaling, force transduction, and regulation of the actin cytoskeleton. Vinculin has at least 10 binding partners distributed throughout the FA including paxillin in the ISL, talin in the FTL, and actin in the ARL. We hypothesize that vinculin interacts with distinct binding partners within distinct FA layers to regulate its activation and mediate its functional specificity. To test this, we utilized point mutants to perturb specific protein interactions and assayed their nanoscale localization, activation state, and binding stability in FAs. Our results suggest a model in which inactive vinculin is recruited to the ISL near the plasma membrane in FA by a weak interaction with paxillin. We speculate that this localization puts vinculin in proximity of multiple ligands that promote activation, which allows a shift to the FTL and ARL where interactions of activated vinculin with talin and actin promote FA stabilization. We are assaying additional vinculin mutants to provide further evidence for this model. A Manuscript describing this work is being prepared for publication Project 3: Active organization of integrins in focal adhesions Vinay Swaminathan, Pontus Norenfeldt, Joseph Matthew, Timothy Springer, Satyajit Mayor Integrins are transmembrane ECM receptors that link the extra-cellular matrix (ECM) and the cytoskeleton and play a crucial role in the immune response, cell migration and tissue morphogenesis. ECM-engaged integrins cluster together with proteins that mediate their signaling functions and linkage to the cytoskeleton to form focal adhesions that grow and turn over in an actin and myosin II dependent manner. How actin and myosin mediate the clustering and organization of integrins during activation, focal adhesion growth and turnover is not known. We utilized fluorescence emission anisotropy imaging of cells expressing GFP-tagged integrins to analyze the evolution of integrin organization during focal adhesion dynamics in migrating fibroblasts and to test the role of integrin activation and actomyosin contractility in this process. By employing specific perturbations we are attempting to understand how the association of focal adhesion components and the acto-myosin machinery affect the organization of integrins during the formation of mature adhesions. Project 4: A novel actin-adhesion structure involved in nuclear positioning requires the formin FMN2 Colleen T. Skau and Clare M. Waterman Active asymmetric positioning of the nucleus in nondividing cells is critical to a variety of cell functions, including cell migration, particularly in complex 3D environments. Based on the importance of integrin-FAK signaling in controlling nuclear orientation in migrating fibroblasts, we hypothesize that adhesion of the cell body to the extracellular matrix is likely critical in nuclear positioning. Furthermore, involvement of LPA/Rho signaling in nuclear orientation suggests a role for the actin cytoskeleton in nuclear positioning. We therefore hypothesize that the actin cytoskeleton, working with adhesions, exerts force on the nucleus through its connections to the nuclear lamina via proteins spanning the nuclear envelope. Although proteins that bind actin at the nuclear envelope have been identified, it is unclear specifically which adhesion/ actin structures mechanically couple the nucleus to the extracellular matrix to control active nuclear positioning. To address this question, we examined the interplay between actin, adhesions and the nucleus in live and fixed mouse embryonic fibroblasts using fluorescence microscopy. We identified novel adhesive structures located underneath the nucleus termed subnuclear adhesions that are compositionally and dynamically distinct from canonical focal adhesions found near the leading edge of migrating cells. Unlike focal adhesions at the leading edge, subnuclear adhesions assemble in bursts toward the front of the cell and translocate along the ventral surface of the cell. Additionally, we find that subnuclear adhesions contain a distinct set of proteins from leading edge adhesions, and that these proteins assemble with distinct kinetics. Subnuclear adhesions, like leading edge adhesions, have a specific set of actin filaments associated with them. These subnuclear actin fibers connect to subnuclear adhesions not leading edge adhesions, and we have found that they physically impinge upon the nucleus and help control nuclear shape. We have previously identified the actin nucleation factor formin FMN2 in a screen of adhesion components. We now find that the formin FMN2 localizes underneath the nucleus and is important for these subnuclear actin fibers, which are likely involved in known active positioning mechanisms of the nucleus in migrating cells. Together, our data reveal a previously unidentified mechanism for control of nuclear position via a novel structure connecting the actin cytoskeleton, which impinges upon the nucleus, to the extracellular matrix underneath the cell body.